US11124843B2

US11124843B2 - Method and kit for detecting bacteria of genus nitrobacter

Info

Publication number: US11124843B2
Application number: US15/563,000
Authority: US
Inventors: Tomokazu MITSUI
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2015-04-03
Filing date: 2016-03-30
Publication date: 2021-09-21
Anticipated expiration: 2036-03-30
Also published as: CN108291263A; JP6501594B2; US20180057858A1; WO2016159183A1; CN108291263B; KR102565490B1; TWI729982B; KR20170133484A; TW201643254A; JP2016195564A

Abstract

Provided is a method for detecting a bacterium of the genus Nitrobacter, comprising: a first step of amplifying a nucleotide using a test DNA as a template and primers capable of amplifying a nucleotide sequence of consecutive 110 nucleotides or more and 157 nucleotides or less in the nucleotide sequence set forth in SEQ ID NO: 1 to obtain an amplified product; and a second step of detecting the amplified product.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Application No. PCT/JP2016/060560, filed Mar. 30, 2016, which was published in the Japanese language on Jun. 10, 2016 under International Publication No. WO 2016/159183 A1, and the disclosure of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “Sequence Listing 688101_0053”, creation date of Sep. 27, 2017, and having a size of 6.1 KB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a method and a kit for detecting a bacterium of the genus Nitrobacter.

BACKGROUND ART

As nitrifying bacteria, ammonia-oxidizing bacteria (nitrite bacteria), which oxidize ammonia to nitrite, and nitrite-oxidizing bacteria (nitrate bacteria), which oxidize nitrite to nitrate, are known. These nitrifying bacteria are responsible for nitrogen cycle reactions in the natural world. Meanwhile, in the industry, the nitrifying bacteria play an important role, for example, in the nitrogen removal in the biological treatment process of drainage, represented by the activated sludge process and are involved in the prevention of eutrophication of natural waters due to the outflow of nitrogenous salts.

Activated sludge used in drainage treatment includes various kinds of microorganisms, among which bacteria of the genus Nitrobacter are a kind of nitrite-oxidizing bacteria, which play an important role in the nitrogen removal. In the nitrogen removal, it is desired to monitor and regulate the amount of bacteria of the genus Nitrobacter in activated sludge according to the amount of nitrogen in drainage and the treatment speed required for the drainage treatment. To that end, it is a problem to rapidly detect and quantify only bacteria of the genus Nitrobacter in the activated sludge in which a plurality of microorganisms is present.

Non Patent Literatures 1 and 2 describe real-time PCR with certain primers and a fluorescently labeled probe for detecting bacteria of the genus Nitrobacter.

CITATION LIST Non Patent Literature

Non Patent Literature 1: David W Graham et. al., The ISME Journal vol. 1 (2007), p. 385-393
Non Patent Literature 2: Joke Geeks et. al., Appl Microbiol Biotechnol. vol. 75 (2007), p. 211-221

SUMMARY OF INVENTION Technical Problem

However, when the present inventors tried the detection of bacteria of the genus Nitrobacter in activated sludge using the primers and fluorescently labeled probes described in Non-Patent Literatures 1 and 2, problems occurred such as those that bacteria other than the bacteria of the genus Nitrobacter were detected or that it was not possible to detect some of the bacteria of the genus Nitrobacter. If such a problem occurs, then the quantifiability of the bacteria of the genus Nitrobacter is also affected and it becomes difficult to control the reactions for removing nitrogen in drainage.

In view of the aforementioned circumstances, an object of the present invention is to provide a method for detecting a bacterium of the genus Nitrobacter, the method being capable of specifically detecting and quantifying the bacterium of the genus Nitrobacter even in systems in which a plurality of kinds of microorganisms is present and a kit to be used therefor.

Solution to Problem

The present inventors cloned a nucleotide sequence (SEQ ID NO: 18) of the 16S rRNA gene of a bacterium of the genus Nitrobacter present in activated sludge and newly found that particular regions are useful for the specific detection and quantification of the bacteria of the genus Nitrobacter in systems in which a plurality of kinds of microorganisms is present, thereby completing the present invention.

Accordingly, the present invention relates to, for example, the following [1] to [6]:

[1] A method for detecting a bacterium in the genus Nitrobacter, comprising: a first step of amplifying a nucleotide using a test DNA as a template and primers capable of amplifying a nucleotide sequence of consecutive 110 nucleotides or more and 157 nucleotides or less in the nucleotide sequence set forth in SEQ ID NO: 1 to obtain an amplified product; and a second step of detecting the amplified product.
[2] The method according to [1], wherein the primers used in the first step are a first primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with a nucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2 under stringent conditions and a second primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with a nucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions.
[3] The method according to [2], wherein the first primer comprises one nucleotide sequence selected from nucleotide sequences set forth in SEQ ID NOs: 4 to 8 and the second primer comprises one nucleotide sequence selected from nucleotide sequences set forth in SEQ ID NO: 9 or 10.
[4] The method according to any one of [1] to [3], wherein the second step comprises detecting the amplified product using a probe comprising the nucleotide sequence set forth in SEQ ID NO: 11.
[5] A kit to be used in detection of a bacterium in the genus Nitrobacter, comprising: a first primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with a nucleotide comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2 under stringent conditions and a second primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions; and a probe comprising the nucleotide sequence set forth in SEQ ID NO: 11.
[6] The kit according to [5], wherein the first primer comprises one nucleotide sequence selected from nucleotide sequences set forth in SEQ ID NOs: 4 to 8 and the second primer comprises one nucleotide sequence selected from nucleotide sequences set forth in SEQ ID NO: 9 or 10.

Advantageous Effects of Invention

According to the present invention, it is possible to provide a method for detecting a bacterium of the genus Nitrobacter, the method being capable of specifically detecting and quantifying the bacterium of the genus Nitrobacter even in systems in which a plurality of kinds of microorganisms is present and a kit to be used therefor. By virtue of this, for example, the reactions for removing nitrogen in drainage can be controlled based on the amount of the bacteria of the genus Nitrobacter detected in activated sludge in drainage treatment.

DESCRIPTION OF EMBODIMENTS

A mode for carrying out the present invention (hereinafter, referred to as the “the present embodiment”) is described in detail below. The present invention is not limited to the following embodiment.

<Method for Detecting Bacteria of Genus Nitrobacter>

A method for detecting a bacterium in the genus Nitrobacter of the present embodiment comprises a first step of amplifying a nucleotide using a test DNA as a template and primers capable of amplifying a nucleotide sequence of consecutive 110 nucleotides or more and 157 nucleotides or less in the nucleotide sequence set forth in SEQ ID NO: 1 to obtain an amplified product; and a second step of detecting the amplified product.

Examples of the test DNA according to the present embodiment include a plasmid, cDNA, genomic DNA, and DNA prepared from activated sludge. The test DNA may comprise DNA derived from a plurality of kinds of microorganisms. As a method for preparing the test DNA, a method well known to those skilled in the art may be used. As the test DNA, DNA prepared from activated sludge is preferred from the point of view of controlling reactions for removing nitrogen in drainage. As a method for preparing DNA from activated sludge, for example, commercially available DNA extraction kits may be used.

[First Step]

SEQ ID NO: 1 sets forth a nucleotide sequence of the positions 901 to 1057 of SEQ ID NO: 18. SEQ ID NO: 18 sets forth a nucleotide sequence of the 16S rRNA gene of a bacterium of the genus Nitrobacter. In the 16S rRNA gene, there are preserved regions containing common sequences between species on the gene and 9 variable regions (V1 to V9) containing sequences that vary depending on the species, the genus, or the like. In SEQ ID NO: 18, a nucleotide sequence of the positions 747 to 774 is the V5 region, a nucleotide sequence of the positions 920 to 966 is the V6 region, and a nucleotide sequence of the positions 1038 to 1074 is the V7 region.

The nucleotide sequence set forth in SEQ ID NO: 1 is a region containing the V6 and V7 regions of the 16S rRNA gene. In the first step according to the present embodiment, the amplified product is prepared by amplifying a nucleotide using primers capable of amplifying a nucleotide sequence of consecutive 110 nucleotides or more and 157 nucleotides or less in the nucleotide sequence set forth in SEQ ID NO: 1. The amplified product may be prepared by amplifying a nucleotide using primers capable of amplifying a nucleotide sequence of consecutive 120 nucleotides or more and 157 nucleotides or less or using primers capable of amplifying a nucleotide sequence of consecutive 125 nucleotides or more and 157 nucleotides or less. The amplified product may be a nucleotide sequence having 1 to 2 nucleotides different from the correspondent region in the nucleotide sequence set forth in SEQ ID NO: 1 or a nucleotide sequence having the same nucleotides as long as it is prepared by the amplification using such primers. By using the aforementioned amplified product, the bacteria of the genus Nitrobacter can be specifically detected in the subsequent second step.

The method for amplifying the nucleotide to obtain an amplified product in the first step is not particularly limited, but a method well-known to those skilled in the art may be used. Examples of such a method include polymerase chain reaction (PCR), real-time PCR, and LAMP. Among these methods, real-time PCR is preferred since, at the same time as the amplification, the detection in the second step may be conducted and the amplified product may be quantified.

The primer used to obtain the amplified product may be designed so as to be capable of amplifying a region containing the part from the V6 region to the V7 region (the nucleotide sequence region of the positions 901 to 1057 of SEQ ID NO: 18) of the 16S rRNA gene of the bacteria of the genus Nitrobacter. The primers are a first primer (forward primer) that hybridizes with the antisense strand of the DNA encoding 16S rRNA under stringent conditions and a second primer (reverse primer) that hybridizes with the sense strand under stringent conditions.

As the first primer, a primer that hybridizes with a nucleotide comprising the nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2 under stringent conditions is preferable. The length of the primer is preferably 18 nucleotides or more from the point of view of obtaining more sufficient specificity. The upper limit of the length of the primer is preferably 28 nucleotides or less, more preferably 27 nucleotides or less, and further preferably 25 nucleotides or less from the point of view of increasing annealing efficiency. Specific examples of the first primer include a primer comprising a nucleotide sequence selected from the nucleotide sequences set forth in SEQ ID NOs: 4 to 8.

Examples of the primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with a nucleotide comprising the nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2 under stringent conditions include a primer comprising a nucleotide sequence having 2 nucleotides different from a nucleotide sequence of consecutive 18 nucleotides or more in the nucleotide sequence set forth in SEQ ID NO: 2, preferably a primer comprising a nucleotide sequence having 1 nucleotide different from a nucleotide sequence of consecutive 18 nucleotides or more in the nucleotide sequence set forth in SEQ ID NO: 2, and more preferably a primer comprising a nucleotide sequence of consecutive 18 nucleotides or more in the nucleotide sequence set forth in SEQ ID NO: 2.

As the second primer, a primer that hybridizes with a nucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions is preferable. The length of the primer is preferably 18 nucleotides or more, more preferably 19 nucleotides or more, and further preferably 20 nucleotides or more from the point of view of obtaining more sufficient specificity. The upper limit of the length of the primer is preferably 28 nucleotides or less, more preferably 27 nucleotides or less, and further preferably 25 nucleotides or less from the point of view of increasing annealing efficiency. Specific examples of the second primer include a primer comprising a nucleotide sequence selected set forth in SEQ ID NO: 9 or 10.

Examples of the primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with a nucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions include a primer comprising a nucleotide sequence having 2 nucleotides different from a nucleotide sequence of consecutive 18 nucleotides or more in the nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3, preferably a primer comprising a nucleotide sequence having 1 nucleotide different from a nucleotide sequence of consecutive 18 nucleotides or more in the nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3, and more preferably a primer comprising a nucleotide sequence of consecutive 18 nucleotides or more in the nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3.

As used herein, “stringent conditions” means conditions under which complementary strands of nucleotide strands having a homology with a target sequence preferentially hybridize to the target sequence and complementary strands of nucleotide strands that do not have such a homology do not substantially hybridize. Stringent conditions are sequence-dependent and vary in a variety of situations. Longer sequences specifically hybridize at higher temperatures. In general, stringent conditions are selected so that the temperature is about 5° C. lower than the thermal melting temperature (Tm) of the particular sequence at the prescribed ionic strength and pH. Tm is a temperature at which 50% of nucleotides complementary to the target sequence hybridize to the target sequence in equilibrium under the prescribed ionic strength, pH, and DNA concentration.

Examples of the “primer comprising a nucleotide sequence that hybridizes under stringent conditions” include primers that hybridize a nucleotide of interest at high ion concentrations [for example, 6×SSC (900 mM sodium chloride, 90 mM sodium citrate) or the like is used.] at a temperature condition of 65° C. to form a DNA-DNA hybrid and this hybrid is maintained even after washing for 30 minutes at low ion concentrations [for example, 0.1×SSC (15 mM sodium chloride, 1.5 mM sodium citrate) or the like is used.] at a temperature condition of 65° C.

[Second Step]

A method for detecting the amplified product in the second step is not particularly limited, but a method well-known to those skilled in the art may be used. Examples of the method for detecting the amplified product include agarose gel electrophoresis, real-time PCR, sequence analysis, and Southern blotting. Among these methods, detection methods using a probe are preferred from the point of view of superior specificity. Examples of the detection methods using a probe include real-time PCR or Southern blotting and, among them, real-time PCR is preferred from the point of view of being capable of conducting the amplification in the first step at the same time and specific detection and superior quantifiability.

As a probe used in the detection, oligonucleotides that hybridize with sense or antisense strands of the amplified product obtained in the first step under stringent conditions are preferable. The length of the probe is preferably 15 nucleotides or more and 30 nucleotides or less, more preferably 15 nucleotides or more and 25 nucleotides or less, and further preferably 15 nucleotides or more and 20 nucleotides or less from the point of view of specifically detecting the amplified product. Specific examples of the probe include probes comprising the nucleotide sequence set forth in SEQ ID NO: 11.

If the aforementioned probe is used in real-time PCR, then the probe is modified with a fluorescent substance at the 5′ end and a quencher, which inhibits the fluorescence that the fluorescent substance emits, at the 3′ end, respectively. Examples of the fluorescent substance include FAM, TET, HEX, TAMRA, and Cyanine5. Examples of the quencher include TAMRA, BHQ1, BHQ2, and BHQ3. The fluorescent substance and quencher used for the modification of the probe may be selected as appropriate according to the excitation wavelength and the measurement wavelength of the real-time PCR apparatus to be used.

The real-time PCR apparatus is not particularly limited, as long as it includes a thermal cycler that can amplify DNA by PCR and a spectrofluorometer for detecting the amplified product. Examples of the real-time PCR apparatus include StepOnePlus (manufactured by Applied Biosystems).

The amplified sample detected in the second step may be quantified. Therefore, the detection method of the present embodiment may involve not only detecting the bacteria of the genus Nitrobacter in the test DNA, but also quantifying the amount of the bacteria of the genus Nitrobacter included in the test DNA. For example, if real-time PCR is used for the detection of the amplified product in the second step, then the amount of the test DNA may be quantified from a standard curve obtained using standard samples. The standard curve may be made based on Ct values indicating the number of cycles with which the amplified product reaches a certain value and the initial amounts of the template.

In the detection method of the present embodiment, if an amplified product is obtained from the test DNA in the first step and it was possible to detect the amplified product in the second step, it can be determined that the DNA derived from the bacteria of the genus Nitrobacter is contained in the test DNA.

<Kit of Bacteria of Genus Nitrobacter>

The kit used for the detection of the bacteria of the genus Nitrobacter of the present embodiment comprises a first primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with the nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 2 under stringent conditions and a second primer comprising a nucleotide sequence of 18 nucleotides or more that hybridizes with the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions; and a probe comprising the nucleotide sequence set forth in SEQ ID NO: 11.

As the first primer, the second primer, and the probe, those similar to a primer or probe used in the aforementioned detection method may be used.

To the kit of the present embodiment, other reagents or the like may be attached as needed besides the first primer, the second primer, and the probe comprising the nucleotide sequence set forth in SEQ ID NO: 11. Examples of the other reagents include a DNA polymerase, a deoxyribonucleotide mixture (dNTP Mix), buffer solutions, sterilized water, and DNA for control.

If buffer solutions are attached to the kit of the present embodiment, examples of the buffer solutions include buffer solutions that are used in common PCR.

EXAMPLES Example 1: Selection of Primer and Probe

(Materials)

In order to amplify a 16S rRNA gene derived from a bacterium of the genus Nitrobacter, primers and probes were designed. Primers and a probe for amplifying the V6-V7 regions of the 16S rRNA and primers and probes for amplifying the V5-V7 regions are illustrated in Table 1 and Table 2, respectively. Moreover, as primers for amplifying the 16S rRNA gene derived from bacteria of the genus Nitrobacter, primers having the nucleotide sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20 described in Non Patent Literature 2 were also used. As template DNA, a plasmid in which a nucleotide sequence (SEQ ID NO: 18) of the 16S rRNA gene derived from a bacterium of the genus Nitrobacter was introduced was used. The template DNA was obtained by preparing an amplified product by PCR using primers having the nucleotide sequences set forth in SEQ ID NOs: 21 and 22 and DNA extracted from the bacterium of the genus Nitrobacter as a template and then introducing the amplified product into a plasmid. The PCR reaction was conducted: (1) at 94° C., for 2 minutes, (2) at 98° C., for 10 seconds, (3) at 60° C. to 40° C., for 30 seconds, and (4) at 68° C., for 1.5 minutes and the steps of (2) to (4) were repeated for 35 cycles.

TABLE 1

Kind	Region	SEQ ID NO:	Name

Forward primers	V6	4	FP1
		5	FP2
		6	FP3
		7	FP4
		8	FP5
Reverse primers	V7	9	RP1
		10	RP2
Fluorescently labeled probe	Between V6-V7	11	Probe 1
(5′ end: 6-FAM modified
3′ end: TAMRA)

TABLE 2

Kind	Region	SEQ ID NO:	Name

Forward primers	V5	12	FP6
		13	FP7
		14	FP8
		15	FP9
Reverse primers	V7	9	RP1
		10	RP2
		16	RP3
Fluorescently labeled probe	Between V6-V7	11	Probe 1
(5′ end: 6-FAM modified		17	Probe 2
3′ end: TAMRA)

(Method)

Using the primers and probes set forth in Tables 1 and 2, the 16S rRNA gene of the bacterium of the genus Nitrobacter was amplified with a real-time PCR apparatus (StepOnePlus, manufactured by Applied Biosystems).

10 μL of 2× TaqMan fast Advanced Master Mix (manufactured by Life technologies), 0.36 μL of a forward primer (50 pmol/μL), 0.36 L of a reverse primer (50 pmol/μL), 0.50 μL of a probe, 6.78 μL of sterilized water, and 2.00 μL of template DNA were added to a microtube and mixed. The microtube was placed in the real-time PCR apparatus and the amplification reaction was conducted. The reaction was conducted (1) at 95° C., for 20 seconds, (2) at 95° C., for 1 second, and (3) at 60° C., for 20 seconds and the steps (2) and (3) were repeated for 50 cycles. The progress of the reaction was confirmed in real time by irradiation with excitation light at 492 nm and measurement of fluorescence (515 nm) of the fluorescent substance 6-FAM.

(Result)

The combinations of primers and a probe with which it was possible to amplify the 16S rRNA gene derived from a bacterium of the genus Nitrobacter are illustrated in Tables 3 and 4. 10 sets of primers and a probe designed within the V6-V7 regions and 11 sets of primers and a probe designed within the V5-V7 regions were those with which it was possible to detect the plasmid in which the nucleotide sequence (SEQ ID NO: 18) of the 16S rRNA gene derived from a bacterium of the genus Nitrobacter was introduced and it was possible to detect 1 pg/μL to 0.001 pg/μL of the plasmid. On the other hand, it was not possible to detect the plasmid in which the 16S rRNA gene having the nucleotide sequence set forth in SEQ ID NO: 18 was introduced with the combination of primers having the nucleotide sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20 and Probe 1.

TABLE 3

	Combinations

V6-V7 region	1-1	1-2	1-3	1-4	1-5	1-6	1-7	1-8	1-9	1-10

Forward primer	FP1	FP2	FP3	FP4	FP5	FP1	FP2	FP3	FP4	FP5
Reverse primer	RP1	RP1	RP1	RP1	RP1	RP2	RP2	RP2	RP2	RP2
Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe
	1	1	1	1	1	1	1	1	1	1

TABLE 4

	Combinations

V5-V7 region	2-1	2-2	2-3	2-4	2-5	2-6	2-7	2-8	2-9	2-10	2-11

Forward primer	FP7	FP8	FP9	FP6	FP7	FP8	FP9	FP6	FP7	FP9	FP6
Reverse primer	RP1	RP1	RP1	RP2	RP2	RP2	RP2	RP2	RP3	RP3	RP2
Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe	Probe
	1	1	1	1	1	1	1	1	1	1	2

Example 2: Specificity Test with 1 Kind of Plasmid

(Materials)

Combination 1-2, 1-4, 1-5, 1-7, 1-9, 1-10, or 2-5 of primers and a probe

Plasmids in which the 16S rRNA genes derived from various bacteria were introduced (derived from the bacteria set forth in Table 5).

(Method)

Real-time PCR was conducted by a method similar to that in Example 1 except that the aforementioned primers and probe were used and that any one of the aforementioned plasmids was used as template DNA.

(Result)

Table 5 illustrates the names of the bacteria genera from which the 16S rRNA genes introduced into the plasmid used for the test are derived and the results of detection of these plasmids. Using the combinations (1-2, 1-4, 1-5, 1-7, 1-9, 1-10) of primers and a probe designed within the V6-V7 regions and the combination (2-5) of primers and a probe designed within the V5-V7 regions, the plasmids in which the 16S rRNA gene derived from a bacterium other than the bacteria of the genus Nitrobacter was introduced were below the detection limit (0.001 pg/μL). Moreover, these sets of primers and a probe exhibited no detectability for bacteria of the genus Bradyrhizobium, which is close species of the bacteria of the genus Nitrobacter. Thus, it was demonstrated that it is possible to specifically detect the bacteria of the genus Nitrobacter with these primers and probe.

TABLE 5

	Theoretical
	value of	Detection result of plasmid
Name of bacterial	plasmid	Combinations of primer and probe

genus	(pg/μL)	1-2	1-4	1-5	1-7	1-9	1-10	2-5

Novosphingobium	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Xanthobacter	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Sediminibacterium	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Rhodococcus	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Methyloversatilis	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Methylibium	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Sphingomonas	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Haliscomenobacter	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Nitrosomonas	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Thermomonas	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Bradyrhizobium	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Rhodoplanes	1	Below	Below	Below	Below	Below	Below	Below
		detection	detection	detection	detection	detection	detection	detection
		limit	limit	limit	limit	limit	limit	limit
Nitrobacter	1	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable
	0.1	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable
	0.01	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable
	0.001	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable	Detectable

Example 3: Specificity Test and Quantifiability Test with Mixed Plasmids of Plurality of Kinds

(Materials)

Combination 1-2, 1-4, 1-5, 1-7, 1-9, 1-10, 2-5 of primers and a probe.

Plasmids in which the nucleotide sequence (SEQ ID NO: 18) of the 16S rRNA gene derived from a bacterium of the genus Nitrobacter is introduced.

(Method)

To a mixture (the final concentrations of the various kinds of plasmids were each 1 pg/μL) in which the plasmids in which the 16S rRNA genes derived from 12 kinds of bacteria other than the bacteria of the genus Nitrobacter among the bacteria set forth in Table 5 were introduced were mixed, a plasmid in which the 16S rRNA gene derived from a bacterium of the genus Nitrobacter was introduced was admixed at a final concentration of 1 pg/μL, 0.1 pg/μL, 0.01 pg/μL, or 0.001 pg/μL to prepare a plasmid mixture. Real-time PCR was conducted by a method similar to that in Example 1 except that the aforementioned combinations of primers and a probe were used and that the prepared plasmid mixtures were used as template DNA.

(Result)

Table 6 illustrated the measurements (pg/μL) of the plasmid in which the 16S rRNA gene derived from the bacterium of the genus Nitrobacter was introduced, with the plasmid quantified by real-time PCR. If the combinations 1-2, 1-4, 1-5, 1-7, 1-9 and 1-10 of primers and a probe with the primers designed within V6-V7 regions were used, it was possible to specifically detect only the plasmid derived from the genus Nitrobacter and to quantify the plasmid at concentrations down to 0.001 pg/μL, even when plasmids in which the 16S rRNA genes derived from a plurality of kinds of bacteria were introduced as template DNA. At this time, the plasmids in which the 16S rRNA gene derived from a bacterium other than the bacteria of the genus Nitrobacter was introduced were below the detection limit (0.001 pg/μL). Meanwhile, if the combination 2-5 of primers and a probe with the primers designed within the V5-V7 regions was used, it was possible to specifically detect only the plasmid derived from the genus Nitrobacter at concentrations down to 1 pg/μL, but it was not possible to detect and quantify the plasmid derived from the genus Nitrobacter at concentrations of 0.1 pg/μL or less.

TABLE 6

			Theoretical values of plasmid (pg/μL)

			1	0.1	0.01	0.001

Measurements	Combi-	1-2	3.3	0.081	0.0085	0.00017
of plasmid	nations	1-4	2.2	0.11	0.011	0.0010
(pg/μL)		1-5	2.5	0.10	0.013	0.00085
		1-7	2.9	0.053	0.0057	0.00085
		1-9	1.7	0.082	0.011	0.00047
		1-10	2.1	0.056	0.0064	0.00069
		2-5	1.4	Below	Below	Below
				detection	detection	detection
				limit	limit	limit

Example 4: Specificity Test with 1 Kind of Genomic DNA

(Materials)

The combination 1-9 of primers and a probe

Genomic DNAs derived from various kinds of bacteria (derived from the bacteria set forth in Table 7)

(Method)

Real-time PCR was conducted by a method similar to that in Example 1 except that the combination 1-9 of primers and a probe was used and that genomic DNAs (1 ng/μL) derived from the bacteria set forth in Table 7 were used as template DNA.

Table 7 illustrates the bacteria from which the genomic DNAs used in the test were derived and the results of detection of their genomic DNAs. Using the combination 1-9 of primers and a probe, it was possible to detect genomic DNA of Nitrobacter winogradskyi NBRC14297, a bacterium of the genus Nitrobacter, at concentrations down to 0.001 ng/μL. Meanwhile, genomic DNAs derived from the other bacteria were below the detection limit (0.001 ng/μL). Therefore, it was demonstrated that it is possible to specifically detect the bacteria of the genus Nitrobacter with this combination of primers and probe even when genomic DNA is used.

TABLE 7

	Theoretical
	value of
Bacterium from which genomic DNA is	genomic DNA	Detection result of
derived	(ng/μL)	genomic DNA

Bacillus licheniformis IFO 12200t	1	Below detection limit
Bacillus subtilis JCM 1465t	1	Below detection limit
Rhizobium galegae IFO 14965t	1	Below detection limit
Rhizobium meliloti IFO 14782t	1	Below detection limit
Rhizobium leguminosarum IFO 14778t	1	Below detection limit
Escherichia coli JM109	1	Below detection limit
Rhodococcus rhodochrous JCM 3202t	1	Below detection limit
Sphingomonas parapaucimobilis JCM 7510t	1	Below detection limit
Sphingomonas paucimobilis IFO 13935t	1	Below detection limit
Sphingomonas yanoikuyae JCM 7371t	1	Below detection limit
Sphingomonas adhaesiva JCM 7370t	1	Below detection limit
Bradyrhizobium diazoefficiens NBRC14792	1	Below detection limit
Bradyrhizobium japonicum NBRC14783	1	Below detection limit
Nitrobacter winogradskyi NBRC14297	1	Detectable
	0.1	Detectable
	0.01	Detectable
	0.001	Detectable

Example 5: Quantifiability Test with Mixed Genomic DNAs of Plurality of Kinds

(Materials)

The combination 1-9 of primers and a probe

Genomic DNA derived from Nitrobacter winogradskyi NBRC4297

Genomic DNAs derived from various kinds of bacteria (derived from the bacteria set forth in Table 8)

(Method)

To a mixture (the final concentrations of the various kinds of genomic DNAs were each 100 pg/μL) in which genomic DNAs derived from the bacteria set forth in Table 8 were mixed, genomic DNA derived from Nitrobacter winogradskyi NBRC14297 was admixed at a final concentration of 100 pg/μL, 10 pg/μL, 1 pg/μL or 0.1 pg/μL to prepare a genomic DNA mixture. Real-time PCR was conducted by a method similar to that in Example 1 except that the combination 1-9 of primers and a probe was used and that the prepared genomic DNA mixtures were used as template DNA.

TABLE 8

	Genomic DNA
Name of bacterium	concentration (pg/μL)

Bacillus licheniformis IFO 12200t	100
Bacillus subtilis JCM 1465t	100
Rhizobium galegae IFO 14965t	100
Escherichia coli JM109	100
Rhodococcus rhodochrous JCM 3202t	100
Sphingomonas parapaucimobilis JCM 7510t	100
Sphingomonas paucimobilis IFO 13935t	100
Sphingomonas yanoikuyae JCM 7371t	100
Sphingomonas adhaesiva JCM 7370t	100

(Result)

Table 9 illustrates the results of quantification of genomic DNA derived from Nitrobacter winogradskyi NBRC14297 with the combination 1-9 of primers and a probe. The measurements of genomic DNAs illustrated in Table 9 are the means of results of measurements of 3 samples. If the combination 1-9 of primers and a probe was used, then it was possible to specifically detect only the genomic DNA derived from Nitrobacter winogradskyi NBRC14297 and to quantify the concentrations down to 0.1 pg/μL even when genomic DNAs derived from a plurality of kinds of bacteria as template DNA. At this time, the genomic DNAs derived from the other kinds of bacteria were below the detection limit (0.1 pg/μL).

TABLE 9

Theoretical value of genomic DNA (pg/μL)	100	10	1	0.1
Measurement of genomic DNA (pg/μL)	71	12	1.7	0.27

Example 6: Detection and Quantification of Bacteria of Genus Nitrobacter with Activated Sludge

(Materials)

The combination 1-9 of primers and a probe

Genomic DNAs derived from activated sludge samples 1-11 (amount of activated sludge sampled was 1.5 mL and amount of elution was 150 μL).

(Method)

Real-time PCR was conducted by a method similar to that in Example 1 except that the combination 1-9 of primers and a probe was used and that genomic DNAs (2 μL) derived from the activated sludge samples 1-11 were used as template DNA.

(Result)

Table 10 illustrates the result of quantification of the genomic DNAs derived from the activated sludge samples 1-11 in terms of the plasmid concentration when the combination 1-9 of primers and a probe was used. It was demonstrated from this result that using the combination 1-9 of primers and a probe, the bacteria of the genus Nitrobacter can be directly detected from genomic DNA derived from activated sludge.

	TABLE 10

	Sample name	Measurement (pg/μL)

	Activated sludge sample 1	0.089
	Activated sludge sample 2	0.088
	Activated sludge sample 3	0.071
	Activated sludge sample 4	0.056
	Activated sludge sample 5	0.073
	Activated sludge sample 6	0.064
	Activated sludge sample 7	0.061
	Activated sludge sample 8	0.100
	Activated sludge sample 9	0.044
	Activated sludge sample 10	0.038
	Activated sludge sample 11	0.036

From these results, it was possible to specifically detect and accurately quantify the bacteria of the genus Nitrobacter by using primers and a probe designed within the V6-V7 regions even in systems in which a plurality of bacterial kinds is present.

Claims

The invention claimed is:

1. A method for detecting a bacterium of the genus Nitrobacter in activated sludge, comprising:

a) obtaining DNA prepared from activated sludge; and

b) detecting whether a bacterium of the genus Nitrobacter is present in the activated sludge sample by:

a first step of amplifying a nucleotide using the DNA prepared from activated sludge as a template and primers capable of amplifying a nucleotide sequence of consecutive 110 nucleotides or more and 157 nucleotides or less in the nucleotide sequence of SEQ ID NO:1 to obtain an amplified product; and

a second step of detecting the amplified product;

wherein the primers used in the first step are a first primer and a second primer, wherein the first primer consists of the nucleotide sequence selected from the group consisting of SEQ ID NO:4 to SEQ ID NO:8 and the second primer consists of the nucleotide sequence selected from the group consisting of SEQ ID NO:9 or SEQ ID NO:10, and

wherein the second step comprises detecting the amplified product using a probe comprising the nucleotide sequence of SEQ ID NO:11, wherein the probe is modified with a fluorescent substance.

2. The method according to claim 1, wherein the first primer consists of the nucleotide sequence of SEQ ID NO:7 and the second primer consists of the nucleotide sequence.